EP1882031B1 - Dendritic cell compositions and methods - Google Patents

Dendritic cell compositions and methods Download PDF

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Publication number
EP1882031B1
EP1882031B1 EP06749566.3A EP06749566A EP1882031B1 EP 1882031 B1 EP1882031 B1 EP 1882031B1 EP 06749566 A EP06749566 A EP 06749566A EP 1882031 B1 EP1882031 B1 EP 1882031B1
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rna
cells
dendritic cells
monocytes
cell
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French (fr)
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EP1882031A2 (en
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Rebecca POGUE
Tamara Monesmith
Irina Tcherepanova
Lois Dinterman
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Merix Bioscience Inc
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Merix Bioscience Inc
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
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Definitions

  • the present invention relates to methods for the production of dendritic cells and related compositions useful in the treatment of disease.
  • a common method for preparing dendritic cells is to collect peripheral blood mononuclear cells (PBMCs) from a subject, and then differentiate the monocytes, which are a small proportion of the PBMCs, into DCs. It was widely believed in order to act as suitable precursors for the in vitro manufacture of dendritic cells, monocytes must be either frozen or cultured soon after isolation from a subject. Accordingly, in previous clinical trials where dendritic cell vaccines were made from monocytes, the PBMCs or monocytes were either cultured at approximately 37°C or frozen within a few hours of the collection of PBMCs from a patient.
  • PBMCs peripheral blood mononuclear cells
  • a commercially viable model for manufacturing DC vaccines is to provide one or a relatively small number of facilities that can manufacture DC vaccines from patient PBMCs or monocytes collected at a clinical site and then shipped to a manufacturing site.
  • PBMCs or monocytes collected at a clinical site and then shipped to a manufacturing site.
  • Freezing fresh monocytes requires additional manipulations at the collection site following leukapheresis, and therefore is not a desirable alternative. Accordingly, there is a need to develop methods for manufacturing DC vaccines using PBMCs or monocytes that have been stored during shipment to a manufacturing facility.
  • the present invention satisfies this need and provides additional advantages as well.
  • the inventors have discovered methods which allow the preparation of dendritic cells and dendritic cell vaccines from monocytes which have be stored at 1-34°C for time periods of 6-96 hours following isolation from a subject.
  • the ability to manufacture DCs from stored monocytes allows greater flexibility and shipment of the monocytes from the site of collection to a manufacturing facility.
  • the inventors have found that the dendritic cell vaccines manufactured from stored monocytes are phenotypically superior to dendritic cells manufactured from fresh monocytes.
  • the dendritic cell vaccines manufactured from stored monocytes have increased levels of costimulatory molecules, such as CD80, CD83 and CD86, as well as higher levels of MHC class I and MHC class II molecules as compared to dendritic cells manufactured from fresh monocytes.
  • the invention provides a method for producing dendritic cells from monocytes, comprising: inducing the differentiation into dendritic cells of monocytes that have been incubated at a temperature of 1°C - 34°C for a period of approximately 6 to 96 hours from the time they are isolated from a subject.
  • the invention provides an antigen-loaded dendritic cell obtainable by the method of the invention for use as a pharmaceutical e.g. as a vaccine, wherein said cells have a steady state ratio of ALOX15 RNA, TLR1 RNA, TLR2 RNA, IL- 10 RNA and/or CD69 RNA to ⁇ -actin RNA or GAPDH RNA of less than 1.0, and/or a steady state ratio of CD52 RNA to ⁇ -actin RNA or GAPDH of greater than 1.0.
  • the invention provides a composition comprising dendritic cell obtainable by the method of the invention, wherein said cells have a steady state ratio of ALOX15 RNA, TLR1 RNA, TLR2 RNA, IL-1 ⁇ RNA and/or CD69 RNA to ⁇ -actin RNA or GAPDH RNA of less than 1.0, and/or a steady state ratio of CD52 RNA to ⁇ -actin RNA or GAPDH of greater than 1.0.
  • the invention provides a vaccine comprising the composition of any of the invention.
  • a cell includes a plurality of cells, including mixtures thereof.
  • antigen is well understood in the art and includes substances which are immunogenic, i.e., immunogens, as well as antigenic epitopes. It will be appreciated that the use of any antigen is envisioned for use in the present invention and thus includes, but is not limited to, a self-antigen (whether normal or disease-related), an infectious antigen (e.g., a microbial antigen, viral antigen, etc.), or some other foreign antigen (e.g., a food component, pollen, etc.).
  • infectious antigen e.g., a microbial antigen, viral antigen, etc.
  • foreign antigen e.g., a food component, pollen, etc.
  • the term includes any of a variety of different formulations of immunogen or antigen.
  • the antigen is from a cancer cell or a pathogen.
  • the cancer cell is a renal cancer cell, a multiple myeloma cell or a melanoma cell.
  • Preferred pathogens are HIV and HCV.
  • the antigen is delivered to the antigen presenting cell (APC) in the form of RNA isolated or derived from a cancer cell or a pathogen. "Derived from” includes, but is not limited recombinant variants of naturally occurring sequences, including fusions to unrelated or related sequences.
  • cancer is meant the abnormal presence of cells which exhibit relatively autonomous growth, so that a cancer cell exhibits an aberrant growth phenotype characterized by a significant loss of cell proliferation control.
  • Cancerous cells can be benign or malignant.
  • the cancer affects cells of the bladder, blood, brain, breast, colon, digestive tract, lung, ovaries, pancreas, prostate gland, or skin.
  • the definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also any cell derived from a cancer cell. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • Cancer includes, but is not limited to, solid tumors, liquid tumors, hematologic malignancies, renal cell cancer, melanoma, breast cancer, prostate cancer, testicular cancer, bladder cancer, ovarian cancer, cervical cancer, stomach cancer, esophageal cancer, pancreatic cancer, lung cancer, neuroblastoma, glioblastoma, retinoblastoma, leukemias, myelomas, lymphomas, hepatoma, adenomas, sarcomas, carcinomas, blastomas, etc.
  • compositions and methods include the recited elements, but not excluding others.
  • Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention. Embodiments defined by each of these transition terms are within the scope of this invention.
  • cytokine refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation.
  • Non-limiting examples of cytokines which may be used alone or in combination in the practice of the present invention include, interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interleukin-1 beta (IL-1 ⁇ ), interferon- ⁇ (IFN ⁇ ), tumor necrosis factor- ⁇ (TNF ⁇ ), prostaglandin E 2 (PGE 2 ), MIP-11, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoie
  • LIF le
  • Cytokines are commercially available from several vendors such as, for example, Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R&D Systems (Minneapolis, MN) and Immunex (Seattle, WA). It is intended, although not always explicitly stated, that molecules having similar biological activity as wild-type or purified cytokines (e.g., recombinantly produced or muteins thereof) are intended to be used within the spirit and scope of the invention.
  • dendritic cells refers to a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissues, Steinman (1991) Ann. Rev. Immunol. 9:271-296 . Dendritic cells constitute the most potent and preferred APCs in the organism. Dendritic cells can be differentiated from monocytes, and possess a distinct phenotype from monocytes. For example, a particular differentiating marker, CD 14 antigen, is not found in dendritic cells but is possessed by monocytes. It has been shown that mature DCs can provide all the signals necessary for T cell activation and proliferation.
  • Immature DCs are capable of capturing antigens by endocytosis, phagocytosis, macropinocytosis or adsorptive pinocytosis and receptor mediated antigen uptake, and are phenotypically CD80 - or CD80 low , CD83- or CD83 low , CD86 low , and have high intracellular concentrations of MHC class II molecules.
  • Mature DCs have a veiled morphology, a lower capacity for endocytosis and are phenotypically CD80 high , CD83 high , CD86 high in comparison to immature DCs.
  • the mature DCs secrete IL-12 p70 polypeptide or protein, and/or secrete significantly reduced levels (0 to 500 pg/ml per million DCs) of IL-10.
  • IL-10 and IL-12 levels can be determined by ELISA of culture supernatants collected at up to 36 hrs post induction of DC maturation from immature DCs. Wierda W.G. et al (2000) Blood 96: 2917 . Ajdary S et al (2000) Infection and Immunity 68: 1760 . See Banchereau and Steinman (1998) Nature 392:245 for a review.
  • an “effective amount” is an amount sufficient to effect beneficial or desired results.
  • An effective amount can be administered in one or more administrations, applications or dosages.
  • expression refers to the processes by which polynucleotides are transcribed into mRNA and/or mRNA is translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA of an appropriate eukaryotic host, expression may include splicing of the mRNA. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
  • a bacterial expression vector or cassette includes a promoter (e.g., lac promoter) and for transcription initiation the Shine-Dalgarno sequence and the start codon AUG (Sambrook et al. (1989) supra).
  • a eukaryotic expression vector or cassette typically includes a heterologous or homologous promoter for RNA polymerase II, a Kozak sequence, the start codon AUG, a termination codon for detachment of the ribosome and a downstream polyadenylation signal.
  • a heterologous or homologous promoter for RNA polymerase II typically includes a heterologous or homologous promoter for RNA polymerase II, a Kozak sequence, the start codon AUG, a termination codon for detachment of the ribosome and a downstream polyadenylation signal.
  • Such vectors can be obtained commercially or assembled by the sequences described in methods known in the art.
  • genetically modified means containing and/or expressing a foreign gene or nucleic acid sequence which in turn, modifies the genotype or phenotype of the cell or its progeny. In other words, it refers to any addition, deletion or disruption to a cell's endogenous nucleotides.
  • isolated means separated from constituents, cellular and otherwise, in which the polynucleotide, peptide, polypeptide, protein, antibody, or fragments thereof, are normally associated with in nature.
  • an isolated polynucleotide is one that is separated from the 5' and 3' sequences with which it is normally associated in the chromosome.
  • a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.
  • a "concentrated”, “separated” or “diluted” polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s) thereof is distinguishable from its naturally occurring counterpart in that the concentration or number of molecules per volume is greater than “concentrated” or less than “separated” than that of its naturally occurring counterpart.
  • a non-naturally occurring polynucleotide is provided as a separate embodiment from the isolated naturally occurring polynucleotide.
  • a protein produced in a bacterial cell is provided as a separate embodiment from the naturally occurring protein isolated from a eukaryotic cell in which it is produced in nature.
  • An isolated mammalian cell is separated from where it is normally found in the body, or is removed from the body. For example, leukocytes collected by leukophereis are "isolated”, and dendritic cells differentiated from monocytes in vitro are "isolated”.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • the proteins encoded by the MHC are known as “MHC molecules” and are classified into Class I and Class II MHC molecules.
  • Class I MHC molecules include membrane heterodimeric proteins made up of an ⁇ chain encoded in the MHC noncovalently linked with the ⁇ 2-microglobulin.
  • Class I MHC molecules are expressed by nearly all nucleated cells and have been shown to function in antigen presentation to CD8 + T cells.
  • Class I molecules include HLA-A, B, and C in humans.
  • Class II MHC molecules also include membrane heterodimeric proteins consisting of noncovalently associated ⁇ and ⁇ chains.
  • Class II MHC molecules are known to function in CD4 + T cells and, in humans, include HLA-DP, DQ, and DR.
  • monocytes By monocytes is meant CD14 + peripheral blood mononuclear cells capable of differentiating into immature dendritic cells in response to GM-CSF and IL-4.
  • Phathogen refers to any disease causing organism or virus, and also to attenuated derivatives thereof.
  • a “pharmaceutical composition” is intended to include the combination of an active agent (such as an antigen-loaded DC) with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • an active agent such as an antigen-loaded DC
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as heat-inactivated serum plus 10% DMSO plus 5% dextrose, phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include adjuvants, stabilizers and preservatives.
  • carriers, stabilizers and adjuvants see Remington's Pharm. Sci. 18th Ed. (Mack Publ. Co., Easton (1990 )).
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to polymeric forms of nucleotides of any length.
  • the polynucleotides may contain deoxyribonucleotides, ribonucleotides, and/or their analogs.
  • Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown.
  • polynucleotide includes, for example, single-stranded, double-stranded and triple helical molecules, a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a nucleic acid molecule of the present invention may also comprise modified nucleic acid molecules.
  • peptide is used in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
  • the subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics.
  • a peptide of three or more amino acids is commonly called an oligopeptide if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or a protein.
  • subject refers to a mammal, including, but not limited to, humans and other primates, rodents, dogs and cats. Preferably, the subject is a human.
  • the inventors have discovered that immature dendritic cells, mature dendritic cells and antigen loaded dendritic cells can be produced from monocytes stored at 1°C - 34°C for approximately 6 to 96 hours following collection of the monocytes from a patient. These results are surprising, as it was commonly believed critical to prepare dendritic cells from freshly isolated monocytes, rather than from monocytes that had been stored at ambient temperatures for a significant length of time. Moreover, monocyte-derived DC vaccines used in clinical trials performed prior to the instant invention have been prepared by culturing monocytes within less than 6 hours following collection from a patient, or by freezing PBMCs shortly after collection, and storing the PBMCs for subsequent thawing and culturing of monocytes.
  • dendritic cells and dendritic cell vaccines are phenotypically superior to the dendritic cells made from fresh monocytes.
  • the method of manufacture and the characteristics of dendritic cell vaccines produced by these methods are particularly relevant to vaccine potency, successful commercialization over a widespread area, and ease of administration.
  • the mature dendritic cells manufactured using the methods of the invention when compared to DCs produced by prior art methods, express increased levels CD80, CD83 and CD86 costimulatory molecules, as well as increased levels of MHC class I and MHC class II molecules, all of which are indicative of DC maturity and potency.
  • mature DCs of the invention are able to induce IL-2 production from memory T cells in an antigen specific fashion.
  • antigen-loaded dendritic cells are typically frozen in DMSO and stored until thawing, washing and resuspending in a DMSO-free pharmaceutically acceptable carrier prior to administration to a patient.
  • the washing step was included in prior DC vaccine clinical trials because it was thought that DMSO has detrimental effects on unfrozen or thawed DCs.
  • the inventors have discovered that DMSO has no noticeable detrimental effect on dendritic cells.
  • there is no need to wash and resuspend the thawed DC vaccine prior to administration. Omitting this step increases the ease of administration and decreases both the risk of contamination and of adverse effects to the DCs due to additional manipulations.
  • the invention provides a method for producing dendritic cells from monocytes, comprising:
  • monocyte refers to a CD14 + leukocyte having the capacity to differentiate into a dendritic cell.
  • the monocyte may be from any mammal, and preferably is a human monocyte.
  • the monocytes can be provided and incubated in compositions such as, but not limited to, blood, blood fractions (e.g., white blood cells (WBCs), buffy coats, peripheral blood mononuclear cells (PBMCs), etc, and as well as in compositions further enriched for monocytes.
  • WBCs white blood cells
  • PBMCs peripheral blood mononuclear cells
  • the monocytes are provided together with other peripheral blood mononuclear cells (PBMCs), for example, as a leukapheresis product.
  • PBMCs peripheral blood mononuclear cells
  • the monocytes are enriched from PBMCs, or isolated directly from peripheral blood. Methods of isolating monocytes or PBMCs containing monocytes are known to those of skill in the art.
  • the monocytes are collected together with other PBMCs by leukapheresis. Methods of leukapheresis are known in the art.
  • PBMCs comprising monocytes are collected from a subject by leukapheresis at a hospital, clinic, doctor's office, etc. Leukapheresis is a procedure by which the white blood cells are removed from a subject's blood, the remainder of which is then transfused back into the subject.
  • the leukapheresis product is typically a blood fraction enriched for PBMCs, with low levels of contaminating red blood cells, granulocytes and platelets.
  • Methods and equipment for performing leukapheresis are well known in the art. See, for example gambrobct.com/Products_&_Services/ for detailed information on leukapheresis.
  • Examples of leukapheresis apparatuses include the COBESpectraTM manufactured by GAMBRO BCT, and the CS3000 Plus Blood Cell Separator manufactured by Baxter Fenwal.
  • Monocytes can be enriched from blood or blood fractions (e.g., PBMCs), during or after the 1°C - 34°C incubation period.
  • PBMCs blood or blood fractions
  • enriching monocytes means a method which increases the proportion of monocytes with respect to other cell types that were present at the start of the method.
  • Methods for enriching monocytes from PBMCs, blood or other blood fractions are known to those of skill in the art and include, but are not limited to elutriation, FACS, panning, magnetic sorting, low density Ficoll gradient centrifugation, and the like.
  • monocytes are enriched from PBMCs by elutriation.
  • monocytes are enriched from PBMCs following the 6-96 hour incubation period by selection for monocytes which adhere to plastic during cell culture.
  • monocytes are enriched by immunomagnetic selection. The immunomagnetic selection may be positive selection to bind monocytes, or may be negative selection, to bind cells which are not monocytes (e.g., T cells, B cells, etc.)
  • monocytes e.g., purified monocytes, enriched monocytes, PBMCs comprising monocytes, etc.
  • monocytes are incubated at a temperature of 1°C - 34°C for a period of approximately 6 to 96 hours from the time they are isolated from a subject.
  • the time at which monocytes or PBMCs containing monocytes are isolated from a subject refers to the time at the completion of the process of removing the cells from the subject. For example, where PBMCs are isolated from a patient over the course of a four hour leukapheresis procedure, the time of isolation would be the time at which collection of PBMCs by leukapheresis ends.
  • the monocytes are incubated for 6 to 96 hours at a temperature of 3°C - 34°C, or 4°C - 32°C or 5°C - 30°C, more preferably at a temperature of 6°C - 28°C, even more preferably at a temperature of 6°C - 27°C, 8°C - 26°C or about 14°C - 24°C.
  • Preferred lower temperature ranges are 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C and 14°C.
  • Preferred upper temperature ranges are 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C and 34°C.
  • the period of incubation is 8 to 72 hours, more preferably 10 to 48 hours, even more preferably 12 to 24 hours, and most preferably, 15 to 22 hours.
  • Other preferred ranges of incubation times include 8 to 48 hours, 10 to 30 hours, 26 to 72 hours and 48 to 80 hours.
  • Preferred lower limits of incubation times can be selected from 6, 7, 8, 10, 12, 14, 16, 20, 22, 24, 26, 28, 30, 36 and 48 hours.
  • Preferred upper limits of incubation times can be selected from 24, 26, 28, 30, 36, 48, 60, 72, 84 and 96 hours.
  • Monocytes in any form may be shipped from a clinical site to a dendritic cell manufacturing site during the 1°C - 34°C incubation period.
  • the monocytes are shipped in a temperature controlled container. Methods of maintaining the temperature of the monocytes between 1°C - 34°C during the incubation period are known to those of skill in the art.
  • the monocytes can be incubated in an incubator or a room at 1°C - 34°C.
  • the monocytes are subjected to some motion (either occasional or continuous) during the incubation period.
  • the motion can be the motion associated with shipping.
  • the cells can be gently rocked or rotated during incubation. While not wishing to be bound by theory, it is thought that the motion may prevent cell damage associated with compaction during settling.
  • the monocytes are incubated without culturing.
  • “without culturing” is meant that during the 6 to 96 hour incubation period, the monocytes are not cultured in a mammalian cell culture medium (including, but not limited to, physiologically appropriate concentrations (e.g., about 1X) of culture mediums such as RPMI, DMEM, X-VIVO 15, AIM-V, StemSpan H2000, and the like) at a temperature of about 36-38°C.
  • monocytes processed by the methods of the invention are incubated at 1°C - 34°C, preferably in blood or blood fractions (e.g., serum, plasma, leukapheresis product (e.g., PBMCs), buffy coat, etc.) saline or biological buffers such as phosphate buffer saline (PBS).
  • blood or blood fractions e.g., serum, plasma, leukapheresis product (e.g., PBMCs), buffy coat, etc.
  • saline or biological buffers such as phosphate buffer saline (PBS).
  • PBS phosphate buffer saline
  • the leukapheresis product containing monocytes is incubated at 1°C - 34°C in the leukapheresis collection container (e.g., a blood collection bag). While the leukapheresis product may be transferred to another container at the beginning or during the incubation period, it is preferable to avoid unnecessary
  • the monocytes can be enriched prior to differentiation step. Manipulations may be performed on the monocytes or PBMCs, etc., during the period of incubation, so long as the manipulations are performed at 1°C - 34°C.
  • PBMCs may be further purified, or monocytes may be enriched from PBMCs during this period of incubation.
  • monocytes can be enriched from PBMCs after the incubation period by culture in a container (preferably a plastic container) and selection for adherent monocytes.
  • the monocytes are induced to differentiate into dendritic cells.
  • monocytes are differentiated into immature dendritic cells and then the immature dendritic cells then can be matured into mature dendritic cells.
  • a variety of methods for differentiating monocytes into dendritic cells, and for maturing the dendritic cells are known to those of skill in the art.
  • monocytes are cultured in a medium comprising a composition that induces the differentiation of monocytes into immature or mature dendritic cells.
  • compositions which induce the differentiation of monocytes into immature dendritic cells are known to those of skill in the art. Such compositions include, but are not limited to, GM-CSF + IL-4; GM-CSF + IL-13; GM-CSF + IL-15; IFN ⁇ ; and GM-CSF + TNF ⁇ .
  • the composition which induces differentiation is GM-CSF + IL-4.
  • the concentrations of GM-CSF and IL-4 may range from about 400 to 2000 U/ml of each cytokine.
  • the concentration of GM-CSF and IL-4 is 500 to 1000 units/ml of each cytokine.
  • the monocytes are contacted with GM-CSF and IL-4 for about 4-7 days, most preferably for about 5-6 days, during which time the monocytes differentiate into immature dendritic cells.
  • the immature dendritic cells can be matured into mature dendritic cells.
  • Methods for maturing dendritic cells are known to those of skill in the art.
  • the immature dendritic cell are matured by contact with a medium comprising GM-CSF, IL-4 and a maturation cocktail (PGE 2 , TNF ⁇ , IL-6 and IL-1 ⁇ ). See for example, Jonuliet et al. (1997) Eur J Immunol 27:3135-3142 .
  • immature dendritic cells are signaled with a first signal, comprising IFN- ⁇ , followed by a second signal comprising CD40L.
  • immature dendritic cells are contacted with PGE 2 , IFN- ⁇ and CD40L, preferably in the presence of GM-CSF and IL-4.
  • the contacting with CD40L is effected upon translation of a recombinant CD40L mRNA within the dendritic cells.
  • the dendritic cell is transiently transfected with an mRNA encoding CD40L or an active fragment thereof.
  • immature dendritic cells are contacted with PGE 2 , TNF ⁇ , and IFN ⁇ , preferably in the presence of GM-CSF and IL-4, to produce mature dendritic cells.
  • the maturity of the dendritic cells can be further increased by transfection, preferably transient transfection, with an RNA encoding CD40L.
  • the dendritic cells are transfected with an RNA encoding CD40L and/or RNA encoding one or more antigens or epitopes of interest.
  • the dendritic cells are loaded with one or more antigens.
  • Antigen loaded dendritic cells are useful as vaccines and for the in vitro stimulation of T cells.
  • Antigens can be loaded into immature or mature dendritic cells. If antigens are loaded into immature dendritic cells, the immature dendritic cells can then be matured by the process of loading itself, or by other maturation methods described herein or alternative maturation methods known to those of skill in the art.
  • the antigen(s) can be loaded as the antigen itself (e.g., proteins, peptides, epitopes, cell lysates, viral particles, etc.) or can be loaded as a nucleic acid(s) encoding antigen(s).
  • the antigen is loaded as a nucleic acid encoding the antigen. More preferably, the nucleic acid is an RNA, most preferably an mRNA. In a preferred embodiment mRNA encoding one or more antigens is cotransfected with mRNA encoding CD40L.
  • the antigen is autologous to the subject, and is used to prepare an antigen loaded autologous DC vaccine for administration to the subject. Methods for loading dendritic cells with peptide and protein antigens, cells, cell or tissue lysates, viruses or viral particles, nucleic acids and the like are known to those of skill in the art.
  • the antigen is loaded by electroporation of a dendritic cell (mature or immature) with a nucleic acid, preferably an mRNA.
  • the dendritic cells are transfected with approximately 0.25 to 4 micrograms RNA per 10 6 dendritic cells, most preferably with about 2 ⁇ g RNA per 10 6 dendritic cells.
  • 1 microgram tumor RNA per million DC is used per transfection.
  • 0.25 to 1.0 ⁇ g each of four RNAs encoding four separate antigens from a pathogen (e.g., HIV) is used per 10 6 dendritic cells.
  • the antigen can be from any source. However, in preferred embodiments, the antigen or antigen(s) are autologous to the subject. By autologous to the subject is meant that the antigen is obtained or derived from the subject.
  • the antigens may be from cancer cells or tumor tissue obtained from a subject.
  • the cancer antigens could be loaded into dendritic cells as cancer cells, cancer cell or tissue lysates, extracts from cancer cells or tissues, purified or cloned components of cancer cells or tissues, total RNA or total mRNA, or selected RNA or mRNA from such cells or tissues, whether present in extracts, purified, amplified, in vitro translated and the like.
  • the antigen may be obtained or derived from a pathogen or pathogen-infected cells present in a subject.
  • the methods of the invention are particularly useful for the treatment or prevention of cancer and pathogen infection.
  • the cancer is renal cell carcinoma, melanoma, breast cancer, chronic lymphocytic leukemia, multiple myeloma, lung cancer, colon cancer, pancreatic cancer, stomach cancer or prostate cancer.
  • pathogen refers to any virus or organism which is involved in the etiology of a disease and also to attenuated derivatives thereof.
  • pathogens include, but are not limited to, bacterial, protozoan, fungal and viral pathogens such as Helicobacter, such as Helicobacter pylori, Salmonella, Shigella, Enterobacter, Campylobacter, various mycobacteria, such as Mycobacterium leprae, Bacillus anthracis, Yersinia pestis, Francisella tularensis, Brucella species, Leptospira interrogans, Staphyloccus, (e.g., S.
  • the pathogen is a viral pathogen, more preferably a retroviral pathogen, and most preferably HIV or HCV.
  • Dendritic cells whether mature or immature, antigen loaded or not, can be frozen in a composition comprising a cryoprotectant.
  • cryoprotectants include, but are not limited to, dimtheylsulfoxide (DMSO), glycerol, ethanol, methanol, acetamide, glycerol monoacetate, propane-diol, polyethylene glycol, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, D-lactose, i-inositol, choline chloride, amino acids, albumin (preferably human serum albumin), polyvinyl pyrrolidone, dextran, sucrose, Ficoll, inorganic salts, and hydroxyethyl starch.
  • DMSO dimtheylsulfoxide
  • glycerol glycerol
  • ethanol ethanol
  • methanol acetamide
  • the cyroprotectant is DMSO.
  • the concentration of DMSO is 2-20%, more preferably 5-15%, and most preferably approximately 10%.
  • the freezing medium may contain one or more polyol compounds derived from carbohydrates, such as glucose, dextrose, sucrose, etc., preferably in a concentration of from 2-30%, more preferably from 5-10%, most preferably 5% dextrose. Methods for freezing dendritic cells are known to those of skill in the art. See, for example U.S. patent application 20040253574 ,
  • the cryoprotectant is dimethylsulfoxide (DMSO).
  • the concentration of DMSO is 5% to 20%. Most preferably, the concentration of DMSO in the composition is approximately 10%.
  • dendritic cells and dendritic cell vaccines made by the methods disclosed herein are capable of surviving in vitro for at least 24 hours post-thaw following freezing in the presence of 5%-20% DMSO and thawing. Because the antigen-loaded dendritic cells of the invention are resistant to DMSO, it is not necessary to wash the cell prior to administering the dendritic cell vaccine. Accordingly, the thawed dendritic cell vaccines are ready for administration to a subject at any time after thawing. Eliminating the washing step reduces the risk of contamination and avoids further manipulations that may damage the dendritic cells.
  • the disclosure provides a method for the administration of an antigen loaded dendritic cell vaccine, comprising thawing a frozen dendritic cell vaccine comprising at least 2% to 20% DMSO, and administering the vaccine to a subject without altering the ratio of dendritic cells to DMSO prior to administration.
  • the concentration of DMSO in the vaccine is approximately 5-20%, and more preferably 10%.
  • an antigen-loaded dendritic cell for the preparation of a frozen medicament for the treatment or prevention of cancer or pathogen infection, wherein the medicament comprises at least 2% DMSO and is ready for administration upon thawing.
  • the methods of the invention allow the production of novel dendritic cells with increased functionality and increased levels of maturity markers.
  • the invention provides mature monocyte derived dendritic cells, wherein the mature dendritic cells have increased levels of one or more of CD80, CD83, CD86, MHC class I molecules, or MHC class II molecules as compared to mature dendritic cells prepared from fresh monocytes.
  • the invention provides a mature monocyte derived dendritic cell, wherein the dendritic cell can elicit antigen-specific IL-2 production from a memory T cell.
  • Methods for measuring IL-2 are known in the art. Cell surface markers and expression of other molecules which are characteristic of memory T cells, and which distinguish them from other types of T cells, are disclosed in Figure 10.35 of Immunobiology, 6th Edition, Eds. Janeway et al., Garland Science Publishing, New York , NY, 2005.
  • memory T cells express high levels of CD44, CD45RO, CD45RA, Bcl-2, IFN ⁇ , CD127 and Ly6C; moderate levels of CD122, and CXCR4; low levels of FasL, and are CD69 and CD25 negative.
  • the invention provides a mature monocyte-derived dendritic cell, wherein the steady state ratio of ALOX15 RNA to either ⁇ -actin RNA or GAPDH RNA in the cell is less that 1.0.
  • the ratio is between 0.2 to 0.7, more preferably between 0.4 to 0.5, and most preferably about 0.45.
  • the invention provides a mature monocyte derived dendritic cell, wherein the steady state ratio of CD52 RNA to ⁇ -actin RNA or GAPDH in the cell is greater than 1.0.
  • the ratio is between 1.2 to 5.0, more preferably between 1.5 to 2.2, or between 1.8 to 1.9, and most preferably the ratio is 1.86.
  • the invention provides a mature monocyte derived dendritic cell, wherein the steady state ratio of TLR1 RNA, TLR2 RNA, IL-1 ⁇ RNA or CD69 RNA to ⁇ -actin RNA or GAPDH RNA in the cell is less than 1.0. Preferably the ratio is between 0.2 to 0.9, and more preferably between 0.5 to 0.8.
  • the human ALOX15 mRNA (SEQ ID NO:1) and allelic variants thereof can be detected using the Affymetrix probes of SEQ ID NOs:2-12.
  • the human IL- ⁇ mRNA (SEQ ID NO: 13) and allelic variants thereof can be detected using the Affymetrix probes of SEQ ID NOs: 14-24.
  • the human TLR1 mRNA (SEQ ID NO:25) and allelic variants thereof can be detected using the Affymetrix probes of SEQ ID NOs:26-36.
  • the human TLR2 mRNA (SEQ ID NO:37) and allelic variants thereof can be detected using the Affymetrix probes of SEQ ID NOs:38-48.
  • the human CD69 mRNA (SEQ ID NO:49) and allelic variants thereof can be detected using the Affymetrix probes of SEQ ID NOs:50-60.
  • the human CD52 mRNA (SEQ ID NO:61) and allelic variants thereof can be detected using the Affymetrix probes of SEQ ID NOs:62-77.
  • the human GAPDH mRNA (SEQ ID NO:78) and allelic variants thereof can be detected using the Affymetrix probes of SEQ ID NOs:79-98.
  • the human ⁇ -actin mRNA (SEQ ID NO: 99) and allelic variants thereof can be detected using Affymetrix probes of SEQ ID NOs:100-119.
  • RNA steady state expression levels can be detected by microarray, preferably using the Affymetrix Human Genome U133 Plus 2.0 Array.
  • hybridization can be performed using the gene specific probes listed in the paragraph above.
  • RNA samples extracted from dendritic cells can be applied to the Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, Calif.) according to the manufacture's instruction (Genechip® Expression Analysis Technical Manual, 2004). Briefly, three micrograms of total RNA spiked with Genechip® Poly-A RNA Control Kit (Affymetrix, Santa Clara, Calif.) are converted to first-strand cDNA using SuperScriptTM II reverse transcriptase.
  • Second-strand cDNA synthesis is followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP.
  • the cRNA products are fragmented to around 100 nucleotides, and hybridized for 16 hours to the microarrays.
  • the microarrays are then washed at low (6xSSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin.
  • Fluorescence is amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
  • the GeneChip® Scanner 3000 (Affymetrix, Santa Clara, Calif.) is used to collect fluorescence signal at 3 ⁇ m resolution after excitation at 570 nm. The average signal from two sequential scans is calculated for each microarray feature of interest. Scanned images were analyzed with Genechip® Operating Software v1.1 (Affymetrix, Santa Clara, Calif.). Preferably, high linear correlation (R 2 >0.95) of 4 control RNAs included in Poly-A RNA Control Kit (Affymetrix, Santa Clara, Calif.) is confirmed as a control for the success of the labeling process.
  • Profile data for all genes, or just the genes of interest are imported into the computer program GeneSpringTM and normalized. Three steps are performed in the normalization step according to the standard method suggested by GeneSpringTM for Affymetrix arrays.
  • the ratio of steady state mRNA of interest (ALOX15, IL-1 ⁇ , TLR1, TLR2, CD69 or CD52 mRNA) to steady state GAPDH or ⁇ -Actin mRNA can then be determined by dividing the normalized expression of the mRNA of interest by the normalized expression of GAPDH or ⁇ -action mRNA.
  • the antigen-loaded dendritic cells of the invention are useful as vaccines in the treatment or prevention of disease or for the activation of T cells, which can then be used in therapy.
  • antigen loaded dendritic cells can be used to elicit an immune response against an antigen. They may be used as vaccines to prevent future infection or disease, or to activate the immune system to treat ongoing disease, such as, but not limited to pathogen infection or cancer.
  • the antigen loaded dendritic cells may be formulated for use as vaccines or pharmaceutical compositions with suitable carriers such as physiological buffers or other injectable liquids. The vaccines or pharmaceutical compositions would be administered in therapeutically effective amounts sufficient to elicit an immune response.
  • the dendritic cells are loaded with an antigen autologous to the subject from which the dendritic cell is derived, and administered to the same subject. See for example, U.S. 5,853,719 , which describes the preparation and uses of antigen loaded dendritic cells and particularly RNA loaded dendritic cells.
  • the dendritic cell may be loaded with an antigen that is not autologous to the intended recipient of the DC therapy. Examples of such antigens include, but are not limited to antigens that are known therapeutic targets, such as telomerase, prostate specific antigen, and other tumor markers, or known antigens from a pathogen.
  • a variety of methods for collecting monocytes and PBMCs comprising monocytes from a subject are known to those of ordinary skill in the art. See for example, gambrobct.com/Products_&_Services/ for detailed information on leukapheresis for the collection PBMCs and elutriation for the purification of monocytes.
  • a leukapheresis product and plasma are collected in separate sterile, disposable, single-use cytopheresis bags is collected using the AutoPBSC (Automated Peripheral Blood Stem Cell) procedure on a Gambro BCT COBE Spectra (Gambro BCT, Lakewood, CO).
  • PBMCs are obtained by collecting blood in a heparinized syringe, dilution in PBS, layering over Histopaque 1077 (Sigma), centrifugation and recovery of PBMCs at the interface. See Woodhead et al. (2000) International Immunol 12:1051-1061 . Additional methods of collecting, purifying or fractionating PBMCs are known to those of ordinary skill in the art.
  • the leukapheresis product is collected in a bag and then transported to the vaccine manufacturing facility in a temperature-monitored shipping container maintained at 1-34°C, preferably at about 6-28°C, and most preferably at 8-26°C.
  • Gel packs such as those disclosed in U.S. patent 4,102807 , which aid in the prevention of temperature changes, can be included in the shipping container.
  • the monocytes can be shipped in an insulated container (e.g., ThermoSafeTM model E65 polyurethane foam insulated container), and packed with ThermoSafeTM U-tek gel packs and gel mats as shown in Figure 1 .
  • an insulated container e.g., ThermoSafeTM model E65 polyurethane foam insulated container
  • ThermoSafeTM U-tek gel packs and gel mats as shown in Figure 1 .
  • a 16 oz U-tek gel mat adjusted to a temperature of -1°C is laid flat in the bottom of the E65 container.
  • Two 16 oz U-tek gel mats (-1°C) are folded and placed between the first gel mat and the short wall of the E65 container.
  • Two 16 oz U-tek gel (adjusted to +18°C) are then placed vertically next to the previous gel mats.
  • a ThermoSafeTM INF3000 transplant container is placed between the gels.
  • the leukapheresis bags are placed in a sealed inner bag (STP711).
  • a device which records the temperature of the inner bag can be used to monitor the temperature during shipment.
  • One such device is the ThermoSafeTM DataLogger.
  • the inner bag is placed in a sealed outer bag (STP710) and then the bags are placed in the INF3000 container.
  • a 16 oz U-tek gel (+18°C) is placed on top of the closed INF300 container, topped with Kraft paper and then a 4" foam plug. The box is then sealed with tape and ready for shipment.
  • the leukapheresis product can be further processed or purified, for example by Ficoll density gradient centrifugation at room temperature in 50 ml conical tubes to separate and concentrate the mononuclear cell fraction that includes dendritic cell precursors (monocytes).
  • the cell concentration and cell viability can be determined.
  • Methods of enriching for monocytes include, but are not limited to, density gradient centrifugation (e.g, dilute Ficoll density gradient centrifugation, dilute Percoll density gradient centrifugation, etc.), elutriation, adherence to plastic, tangential flow filtration, fluorescence activated cell sorting (FACS), immunological cell separation techniques (antibody panning to select monocytes or to remove non-monocytes (e.g., leukocytes, macrophages, granulocytes, etc), differential lysis, magnetic cell sorting, etc.), culture in plastic culture bags coated with plastic microcarrier beads, etc. See, for example, O'Doherty et al.
  • magnetic cell sorting can be used to enrich form monocytes by positive selection (CD14+ cells) or by negative selection (i.e., removal of cells that are not monocytes; e.g., CD3+, CD19+ and CD2+ cells).
  • monocytes are enriched from the leukapheresis product by elutriation, an automated method to isolate monocytes from the subject leukapheresis.
  • Methods of leukapheresis are known in the art.
  • elutriation can be performed on the Gambro BCT ElutraTM Cell Separation System (Gambro BCT, Lakewood, CO).
  • Elutriation buffer can be prepared by adding 1000 mL of 5% Human Albumin Serum (HSA) to a 4L bag of Hank's Balanced Salt Solution (HBSS). The cells can be fractionated by elutriation according to the manufacturer's protocol.
  • HSA Human Albumin Serum
  • HBSS Hank's Balanced Salt Solution
  • a modified version of the manufacturer's (Gambro) protocol is used for elutriation, where the final rotor off fraction is the fourth fraction instead of the fifth fraction.
  • CBC with differential analysis can performed on the monocyte fraction to verify purity and recovery. Alternatively, monocyte purity can be assessed by immunophenotyping with CD14.
  • the enriched monocytes can then be differentiated into dendritic cells, or can be frozen and stored for later use. In one embodiment, the cells are frozen in 25 ml or 50 mL freezing bags. Examples of freezing bags include CryocyteTM freezing bags, OrigenTM freezing bags (Cryostore) and PallTM freezing bags.
  • each freezing bag contains 15 mL of up to 3 x 10 9 cells in culture medium (e.g., AIM V, X-VIVO, RPMI, etc.) with approximately 10-12% DMSO and 10-20% heat inactivated, filtered plasma, about 107 to 507 mg/L final concentration of CaCl 2 and about 44 to 241 mg/L final concentration of MgSO 4
  • culture medium e.g., AIM V, X-VIVO, RPMI, etc.
  • the cells can be frozen using a controlled rate freezer, then stored cryogenically.
  • the PBMCs are resuspended in AIM-V® medium and seeded in T150 cm 2 flasks at 2.0 x 10 8 cells per flask.
  • the PBMCs may be frozen and combined with a second leukapheresis.
  • Monocytes are selected from the mononuclear cell population of PBMCs by adhesion to sterile tissue culture plastic flasks for one to two hours at 37 °C, 5% CO 2 , ⁇ 75% humidity. Non-adherent and semi adherent cells are removed. PBS is added to the flasks to remove the remaining non-adherent cells, semi-adherent cells and residual medium. The remaining adherent cells are predominantly monocytes, and represent a population of enriched monocytes.
  • Examples of alternative methods for differentiating monocytes into dendritic cells include, but are not limited to exposure to physical perturbation (e.g., shearing), irradiation in the presence of a photo-activatable agent capable of forming photoadducts with cellular DNA components, and/or treatment with a DNA binding agent, followed by incubation with disease effector agents, such as microbes, fungi, viruses, and malignant cells. See U.S. patent 6,607,722 .
  • monocytes are differentiated into dendritic cells by culture in medium comprising a composition that induces differentiation of monocytes into dendritic cells.
  • Suitable media for the culture of monocytes, immature and mature dendritic cells includes, but is not limited to, AIM-V, X-VIVO-15, RPMI, DMEM, and the like.
  • Compositions that induce the differentiation of monocytes into dendritic cells are known in the art, and include, but are not limited to, GM-CSF plus IL-4; GM-CSF plus IL-13; and IFN ⁇ .
  • enriched monocytes are differentiated into dendritic cells by culture in the presence of GM-CSF and IL-4 (see, e.g., WO 97/29182 ; Sallusto and Lanzavecchia (1994) J. Exp. Med. 179:1109 ; and Romani et al. (1994) J. Exp. Med. 180:83-93 ).
  • enriched monocytes preferably at a concentration of 1 x 10 6 cells/ml are cultured in AIM V medium, X-VIVO 15 medium, or other suitable medium in the presence 800 U/ml GM-CSF and 500 U/ml IL-4 for approximately 4-7 days, preferably 6 days at 37°C, 5% CO 2 , ⁇ 75% humidity to allow the differentiation of monocytes into immature dendritic cells.
  • Cytokine concentrations can be varied.
  • preferred concentrations of GM-CSF are 500 to 1500U/ml, more preferably 700 to 1000 U/ml, most preferably 800 U/ml.
  • Preferred concentrations of IL-4 are 400-1500 U/ml, more preferably 450 to 1000 U/ml, most preferably 500 U/ml.
  • IL-13 or IL-15 can be used in place of or in addition to IL-4.
  • IFN ⁇ can be used in place of GM-CSF plus IL-4, IL-13 or IL-15.
  • compositions that induce maturation of immature dendritic cells include, but are not limited to, monocyte conditioned medium; PBMC conditioned medium; fixed Staphylococcus aureus (PansorbinTM); lipopolysacharrides (LPS); other bacterial cell products, such as monophosphoryllipid A (MPL), lipoteichoic acid, etc.; phosphorylcholine; calcium ionophores; phorbol esters such as PMA; heat-shock proteins; nucleotides, such as ATP, etc.; lipopeptides; Toll-like receptor 4; artificial ligands for Toll-like receptors; double stranded RNA, such as poly-I:C, etc.; immunostimulant DNA sequences; maturation cocktail (TNF- ⁇
  • a maturation cocktail containing TNF ⁇ , IL-6, IL-1 ⁇ and PGE 2 is added to a culture of immature dendritic cells. The cells are then cultured overnight (approximately 12 hours or more) to produce mature dendritic cells.
  • immature dendritic cells are transfected, preferably by electroporation, with mRNA encoding CD40L, and optionally with mRNA encoding one or more antigens, and then cultured overnight (approximately 12 hours or more) in the presence of IFN ⁇ and optionally PGE 2 to produce mature dendritic cells.
  • a human CD40L cDNA and protein are shown in SEQ ID NO:120 and SEQ ID NO:121, respectively.
  • Other CD40L mRNAs are known to those of skill in the art.
  • a maturation formulation in AIM V medium is added directly to the immature DC to give a final concentration of TNF- ⁇ (10 ng/ml), IFN- ⁇ (1000 U/ml), and PGE 2 (1 ⁇ g/ml).
  • the cells can then cultured overnight (approximately 12 hours or more) to produce mature dendritic cells. Maturation can optionally be further increased by exposure of the cells to CD40 Ligand (CD40L), either added to the culture media, or more preferably expressed within the cell.
  • CD40L can be expressed constitutively or transiently.
  • the mature dendritic cells are transfected with an mRNA encoding CD40L, and optionally with mRNA encoding one or more antigens of interest.
  • antigens can be loaded into immature or mature dendritic cells.
  • the antigen will then be processed and presented by the mature DCs.
  • antigens include, but are not limited to, viral particles, bacteria, or other pathogens, proteins, and fragments thereof, polypeptides, pathogen lysates, pathogen extracts, pathogen nucleic acids, cancer cells, cancer cell proteins and fragments thereof, cancer cell lysates, cancer cell extracts and cancer cell nucleic acids.
  • Antigens can be naturally occurring, chemically processed or recombinantly produced.
  • the antigens can be delivered to the cells as polypeptides, proteins or as nucleic acids using methods known in the art.
  • an antigen may be delivered in its "natural” form in that no human intervention was involved in preparing the antigen or inducing it to enter the environment in which it encounters the dendritic cell.
  • the antigen may comprise a crude preparation, for example of the type that is commonly administered in a conventional allergy shot or in a tumor lysate.
  • the antigen may alternatively be substantially purified, e.g., at least about 90% pure.
  • the antigen is a peptide
  • it may be generated, for example, by proteolytic cleavage of isolated proteins. Any of a variety of cleavage agents may be utilized including, but not limited to, pepsin, cyanogen bromide, trypsin, chymotrypsin, etc.
  • cleavage agents including, but not limited to, pepsin, cyanogen bromide, trypsin, chymotrypsin, etc.
  • peptides may be chemically synthesized, preferably on an automated synthesizer such as is available in the art, or recombinantly expressed.
  • recombinant techniques may be employed to create a nucleic acid encoding the peptide of interest, and to express that peptide under desired conditions.
  • antigen encoding nucleic acids may be purified or derived from a cell, tissue or virus.
  • the antigen can have a structure that is distinct from any naturally-occurring compound.
  • the antigen is a "modified antigen" in that the antigen has a structure that is substantially identical to that of a naturally-occurring antigen but that includes one or more deviations from the precise structure of the naturally-occurring compound.
  • a modified antigen as compared with that protein or polypeptide antigen would have an amino acid sequence that differs from that of the naturally-occurring antigen in the addition, substitution, or deletion of one or more amino acids, and/or would include one or more amino acids that differ from the corresponding amino acid in the naturally-occurring antigen by the addition, substitution, or deletion of one or more chemical moieties covalently linked to the amino acid.
  • the naturally-occurring and modified antigens share at least one region of at least 5 amino acids that are at least 75% identical.
  • Naturally-occurring and modified protein or polypeptide antigens can show at least approximately 80% identity, more alternatively 85%, 90%, 95%, or greater than 99% identity in amino acid sequence for at least one region of at least 5 amino acids. Often, it may be useful for a much longer region (e.g., 10, 20, 50, or 100 or more amino acids) of amino acid sequence to show the designated degree of identity.
  • the antigen is delivered as a polynucleotide or gene encoding the antigen, so that expression of the gene results in antigen production either in the individual being treated (when delivered in vivo) or the cell culture system (when delivered in vitro).
  • Techniques for generating nucleic acids including an expressible gene or mRNA, and for introducing such nucleic acids into an expression system in which any protein encoded by the expressible gene will be produced are known in the art and briefly described infra.
  • the antigen is delivered as an mRNA.
  • RNA or mRNA obtained from a cell for example a cancer cell, pathogen cell or pathogen-infected cell
  • RNA or mRNA can be amplified prior to loading.
  • total or targeted mRNA is amplified by RT-PCR using a primer containing a sense promoter to make a cDNA expression construct.
  • RNA transcribed in vitro from the expression construct can then be used to load the cells.
  • Methods for isolating, amplifying, in vitro transcribing the RNA and loading RNA or other nucleic acids into dendritic cells are known to those of skill in the art.
  • the antigen is one or more HIV proteins or fragments thereof.
  • plasma from an HIV infected patient can serve as a source for isolation of HIV RNA.
  • a portion of the plasma is centrifuged, and the supernatant is collected and filtered using 0.22 ⁇ m filters and stored at - 20°C until use in formulation of the dendritic cell vaccine.
  • the HIV RNA present in plasma is amplified by RT-PCR and in vitro transcription reactions to provide a sufficient quantity of amplified HIV RNA for loading into dendritic cells.
  • viral RNA is reverse transcribed in to single-stranded (ss) DNA using a reverse transcriptase, appropriate reaction buffers and random hexamers or targeted reverse primers.
  • the single-stranded cDNA is then amplified by PCR into double-stranded DNA in a primary PCR reaction using multiplex primers.
  • the identity of region(s) amplified in the primary PCR reaction is determined by the selection of specific primers complimentary to target sequences which flank those regions.
  • the product of the primary PCR reaction is purified using a QIAquick® PCR Purification Kit and then serves as the template in a second round or nested PCR amplification.
  • the 5' primers(s) contains an overhang with an RNA polymerase binding site (e.g., a T7 promoter), and the 3' primer contains an overhang with poly T stretches.
  • the modifications introduced by the overhanging regions in a nested round of PCR enable transcription of the PCR product in vitro and successful translation upon delivery into dendritic cells. Purification of the in vitro transcribed RNA is performed using the Qiagen RNeasy® Kit, and the RNA is eluted in nuclease-free water. If necessary, ethanol precipitation is performed to concentrate the RNA.
  • RNA is re-suspended in nuclease-free water and passed through a 0.8/0.2 ⁇ m polyethersulfone (PES) filter, then dispensed into 0.5 ml safe-lock polypropylene tubes and loaded into DC or cryopreserved at ⁇ -150°C for until thawing prior to transfection.
  • PES polyethersulfone
  • RNA or mRNA is extracted from one or more cancer cells.
  • Dendritic cells may be loaded with one or more antigens as immature dendritic cells, mature dendritic cells, or during differentiation from immature to mature dendritic cells.
  • Dendritic cells are capable of ingesting antigens, such as proteins, peptides, viruses, cells, cell lysates, and the like. Accordingly, antigen loading can be performed simply by contacting the dendritic cell with the antigen or nucleic acid encoding the antigen.
  • Other methods for loading dendritic cells are known to those of skill in the art, including, but not limited to nucleic acid transfection, exosomes, viral vectors, microparticle delivery, etc.. See for example, Mitchell et al.
  • One or more antigens may be loaded directly into the dendritic cells, or nucleic acids encoding one or more antigens may be loaded (transfected) into the dendritic cells.
  • the dendritic cells are loaded with nucleic acids encoding one or more antigens.
  • the nucleic acid is an mRNA.
  • Method of transfecting nucleic acids into dendritic cells include, but are not limited to, passive transfection, lipid-mediated transfection, cationic lipid-mediated transfection (e.g., DOTAP), cationic peptide mediated transfection, electroporation.
  • lipid-mediated transfection e.g., DOTAP
  • cationic lipid-mediated transfection e.g., DOTAP
  • electroporation e.g., electroporation.
  • dendritic cells with proteins, polypeptides, peptides, cell or tissue extracts and other types of antigens are known to those of ordinary skill in the art.
  • immature dendritic cells are loaded with one or more antigens.
  • peptides, polypeptides and/or cell or tissue extracts are loaded simply by incubation with immature dendritic cells in culture medium.
  • immature dendritic cells are harvested by tapping the culture flask to dislodge the cells. Cells in suspension are then transferred to conical tubes. PBS is added to the culture flask to remove the remaining floating cells and residual medium, which is added to the conical flask. Some immature dendritic cells may remain adherent to the flask. Detachment of these cells is promoted by adding PBS and incubating the flasks at anywhere from 2°C up to room temperature. At the end of the incubation period, the flasks are tapped and the dislodged cells are added to the conical tubes.
  • the total cell suspension is then pelleted, washed in PBS and re-suspended in chilled ViaSpan® at 4x10 7 /ml in 0.5ml and placed on ice.
  • DCs are mixed with mRNA at 2 ⁇ g/10 6 cells for mRNA encoding antigen(s) and placed in a 4mm gap electroporation cuvette and electroporated at a pulse of 275-350V, 100-300 ⁇ and 150 ⁇ F, and preferably at 325V, 200 ⁇ .
  • DCs are washed in X-VIVO 15 medium and re-suspended at 1x10 6 /ml in X-VIVO 15 supplemented with GM-CSF (800U/ml), IL-4 (500U/ml) and PGE 2 (1 ⁇ g/ml), TNF- ⁇ (10ng/ml), IL-1 ⁇ (10ng/ml) and IL-6 (100ng/ml).
  • the immature dendritic cells are then incubated overnight at 37°C, 5% CO 2 , ⁇ 75% humidity to produce stably mature dendritic cells. Mature dendritic cells are then washed in PBS.
  • immature dendritic cells are matured using CD40L and IFN- ⁇ .
  • immature DCs are transfected with 4 ⁇ g CD40L mRNA per 10 6 and mRNA (2 ⁇ g/106 cells) encoding one or more antigens by electroporation as described above, and then are cultured overnight in X-VIVO 15 supplemented with GM-CSF (800-1000 U/ml), IL-4 (500-1000 U/ml), IFN- ⁇ (500-1000U/ml) or TNF- ⁇ (10ng/ml) and PGE 2 (1 ⁇ g/ml) to generate stable mature dendritic cells.
  • Dendritic cells matured by this process secrete higher levels of IL-12 (a T cell growth factor), and minimal IL-10, as compared to dendritic cell matured by the cytokine cocktail process described above.
  • Mature Dendritic cells can be loaded with antigen by methods known to those of skill in the art.
  • a maturation formulation in AIM V medium is added directly to the immature DC to give a final concentration of TNF- ⁇ (10 ng/ml), IFN- ⁇ (1000 U/ml), and PGE 2 (1 ⁇ g/ml).
  • the cells are then cultured overnight to produce mature dendritic cells.
  • the DC are then harvested and co-electroporated with 1 ⁇ g of antigen encoding RNA and optionally with 4 ⁇ g of CD40L RNA per 10 6 cells.
  • the cells are cultured for 4 hours at 1 x 10 6 cells AIM V medium supplemented with GM-CSF (800 U/mL), and IL-4 (500 U/mL).
  • the cells can then be formulated for administration to a subject without freezing, or formulated for freezing.
  • the cells are preferably formulated in heat inactivated autologous plasma, 10% DMSO, and 5% dextrose at 2 x 10 7 cells/mL.
  • Cryogenic vials are filled with 0.7 mL for a total number of 1.4 x 10 7 cells per vial.
  • Vials are then frozen in alcohol boxes at -85 °C for a minimum of 4 hours and transferred to the cryogenic freezer for storage.
  • the frozen dendritic cell vaccine can then be thawed and administered to a subject without washing or reformulation.
  • DCs are harvested and re-suspended in chilled PBS/ 1%FCS.
  • Phycoerythrin (PE) or FITC conjugated antibodies specific for MHC molecules (HLA-ABC, HLA-DR), co-stimulatory molecules (CD80, CD86), maturation markers (CD83) and monocyte markers (CD14) are mixed with 1x10 5 DCs per well in a 96 well plates (BD Biosciences) and incubated at 4°C for a minimum of 15 minutes. Isotype matched antibodies were used as controls. After thorough washing, fluorescence analysis was performed with a FACScalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences).
  • the mature dendritic cells are washed and resuspended in heat-inactivated plasma (preferably autologous plasma) and 10% dextrose at a concentration of 4 x 10 7 cells/ml.
  • the cells can then be diluted 1:1 with a mixture of heat-inactivated plasma and 20% DMSO to give a final concentration of 5% dextrose, 10% DMSO in heat-inactivated plasma.
  • the target final filled formulation is 1.4 x 10 7 cells/0.7 ml in a container suitable for cyropreservation.
  • the dendritic cells can then be administered to a patient or frozen, preferably at -85°C, and stored in cryogenic freezer (preferably in a dry liquid nitrogen freezer designed to prevent contamination), preferably at a temperature of ⁇ -150°C.
  • cryogenic freezer preferably in a dry liquid nitrogen freezer designed to prevent contamination
  • the frozen vaccine can then be shipped to a clinical site for patient administration (preferably by intradermal injection). Upon thawing, the vaccine can be administered directly to the patient without further processing.
  • Suitable formulations for administration can include aqueous isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, immunostimulants, cytokines and adjuvants.
  • aqueous isotonic sterile injection solutions which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient
  • aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, preservatives, immunostimulants, cytokines and adjuvants.
  • mature dendritic cells are suspended in heat-inactivated autologous plasma and 10% dextrose, at a final concentration of 4 x 10 7 cells/ml. These cells are then diluted 1:1 with a mixture of heat-inactivated autologous plasma and 20% DMSO to give a final concentration of 2 x 10 7 cells/ml in heat inactivated autologous plasma which contains 5% dextrose and 10% DMSO. The final filled formulation is 1.4 x 10 7 cells/0.7 ml in a container suitable for cyropreservation. The vaccine is then frozen and stored at ⁇ -150 °C in a dry liquid nitrogen freezer. The vaccine is ready for administration after thawing, without the need for washing and resuspending.
  • the dendritic cell vaccine can be administered by a variety of methods, such as, but not limited to, injection (e.g., subcutaneous, intradermal, intravenous, intralymphatic, intraarticular, intramuscular, intraperitoneal), by continuous infusion, sustained release from implants, etc.
  • DC vaccines have typically been administered at two to four week intervals.
  • the dendritic cell vaccine can be administered with physiologically acceptable carriers, buffers, dilutents, adjuvants, immunomodulaters, etc.
  • the dendritic cell vaccine is autologous to the patient it is administered to, or is HLA matched.
  • the dose of cells (e.g., activated T cells, or dendritic cells) administered to a subject is in an effective amount, effective to achieve the desired beneficial therapeutic response in the subject over time, or to inhibit growth of cancer cells, or to inhibit infection.
  • a preferred dose is approximately 10 7 cells.
  • Biological response modifiers are optionally added for treatment by the DCs or activated T cells of the invention.
  • the cells are optionally administered with an adjuvant, or cytokine such as GM-CSF, IL-12 or IL-2.
  • the immunogenicity of the antigen-loaded dendritic cells or educated T cells produced by the methods of the invention can be determined by well known methodologies including, but not limited to the following:
  • Cell isolation or immunoassays for detection of cells during cell purification can be performed in any of several configurations, e.g., those reviewed in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, F la.; Tijan (1985) "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterd am; Harlow and Lane, supra; Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press, Orlando, F la.; Price and Newman (eds.) (1991) Principles and Practice of Immunoassays Stockton Press, NY ; and Ngo (ed.) (1988) Non-isotopic Immunoassays Plenum Press, NY .
  • Cells can be isolated and characterized by flow cytometry methods such a FACS analysis.
  • flow cytometry methods A wide variety of flow-cytometry methods are known.
  • fluorescence activated flow cytometry see, for example, Abbas et al. (1991) Cellular and Molecular immunology W.B. Saunders Company, particularly chapter 3 , and Kuby (1992) Immunology W.H. Freeman and Company, particularly chapter 6.
  • FACS machines are available, e.g., from Becton Dickinson .
  • Labeling agents which can be used to label cell antigen include, but are not limited to monoclonal antibodies, polyclonal antibodies, proteins, or other polymers such as affinity matrices, carbohydrates or lipids. Detection proceeds by any known method, such as immunoblotting, western blot analysis, tracking of radioactive or bioluminescent markers, capillary electrophoresis, or other methods which track a molecule based upon size, charge or affinity.
  • PBMCs Peripheral blood mononuclear cells
  • mononuclear cells were resuspended in StemSpanTM H2000 medium (StemCell Technologies, Inc.) and seeded in T150 cm 2 flasks at 2.0 x 10 8 cells per flask. Monocytes were then selected from the mononuclear cell population by adhesion to the sterile tissue culture plastic flask for 1-2 hours at 37°C, 5% CO 2 , ⁇ 75% humidity. Non-adherent and semi-adherent cells (primarily lymphocytes) were discarded.
  • the remaining adherent cells which were predominantly monocytes, were cultured in StemSpanTM H2000 medium containing GM-CSF (800 U/ml) and IL-4 (500 U/ml). These cells were incubated for 6 days at 37°C, 5% CO 2 , ⁇ 75% humidity to allow the differentiation of monocytes into immature dendritic cells.
  • the immature dendritic cell-rich population was harvested by gently tapping the flasks to dislodge the cells. Cells in suspension were transferred to conical tubes. Additional PBS was added to the flasks to remove the remaining floating cells and residual media and added to the cell suspension in the conical tubes. Detachment of the remaining adherent cells was completed by adding PBS and incubating the flasks at 2-8°C for approximately 10 minutes. While these cells were incubating, the cell suspension in the conical tubes was centrifuged, and the cell pellet was resuspended in PBS. At the end of the incubation period, the flasks were gently tapped and the contents added to the cell suspension in the conical tubes.
  • the total cell suspension was pelleted and resuspended in PBS, and a sample was removed for cell concentration, cell viability and immunophenotyping.
  • the following four sets of cell markers were examined by flow cytometry: monocyte lineage markers (CD3, CD14, CD19, and CD56), indication of presence of dendritic cells (CD11c), an antigen presenting cell marker (HLA-DR), and a mature dendritic cell marker (CD83).
  • the immature dendritic cell-enriched preparation expressed insignificant levels of lineage markers and CD83, and high levels of CD11c and HLA-DR.
  • Immature dendritic cells were washed once with OPTI-MEM® I reduced serum medium (GIBCOTM) with HEPES buffer, L-glutamine, without phenol red.
  • the dendritic cells were then transfected with amplified tumor RNA by electroporation at a ratio of approximately 2 ⁇ g of RNA per 10 6 dendritic cells. Electroporation was performed in 4 mm gap cuvettes containing 600 ⁇ L of a cell suspension containing 5 x 10 7 cells/ml, at a pulse of 500 V for 500 ⁇ s.
  • transfected cells were transferred into T150 flasks (one cuvette per flask) containing StemSpan H2000TM medium (serum free culture medium), supplemented with IL-4 (500 U/ml) and GM-CSF (800 U/ml). Transfected cells were incubated at for 2-3 hours at 37°C, 5% CO 2 , ⁇ 75% humidity to allow the cells to recover from electroporation.
  • StemSpan H2000TM medium serum free culture medium
  • IL-4 500 U/ml
  • GM-CSF 800 U/ml
  • the immature electroporated dendritic cells were matured in StemSpan H2000TM medium, supplemented with IL-4 (500 U/ml) and GM-CSF (800 U/ml) and a maturation cocktail (IL-1 ⁇ at 5ng/ml, IL-6 at 150 ng/ml, TNF- ⁇ at 5 ng/ml and PGE 2 at 1 ⁇ g/ml) at 37°C, 5% CO 2 , ⁇ 75% humidity for 20-24 hours. All cytokines, as well as PGE 2 , were reconstituted or diluted (in the case of PGE 2 ) in PBS with 1% HSA. Prior to the dilution step, PGE 2 was reconstituted in ethanol.
  • Transfected mature dendritic cells were suspended in autologous plasma at a final concentration of 2 x 10 7 cells/ml. The cells were then diluted 1:1 with a mixture of 80% plasma and 20% DMSO to give a final concentrations of 3 x 10 6 or 1 x 10 7 cells/ml in 90% plasma with 10% DMSO, then frozen in cryovials using controlled-rate freezing, and stored at ⁇ -150°C.
  • Dendritic Cell Vaccines Prepared from Day-Old Leukapheresis Product Remain Viable For At Least 2 Hours Post Thaw in 10% DMSO
  • a frozen dendritic cell vaccine prepared as described in Example 1 was thawed at 37°C, and kept at 20-25°C or at 2-8°C for 2 hours. Viability was determined immediately post-thaw and at 30 minute intervals for up to two hours. Viability immediately post-thaw at 37°C was 92%. The results are shown in Table 2, and confirm that the vaccine can be thawed and stored in 10% DMSO for at least two hours. Table 2 Post-thaw time (minutes) Viability (%) 20-25°C Post-thaw 2-8°C Post-thaw 30 91 88 60 89 86 90 89 80 120 87 83
  • Peripheral blood mononuclear cells and plasma were collected from a patient or volunteer by leukapheresis at room temperature at a clinical site.
  • the leukapheresis product (PBMCs) and serum were shipped overnight in a temperature controlled container maintained in a temperature range of 6-28°C.
  • the day following leukapheresis the PBMCs were purified by Ficoll density gradient centrifugation in 50 ml conical tubes at room temperature to separate and concentrate the mononuclear cell fraction that includes monocytes (the dendritic cell precursors) and leukocytes.
  • the mononuclear cells were washed several times in phosphate buffered saline (PBS), and the cell concentration was determined.
  • PBS phosphate buffered saline
  • mononuclear cells were resuspended in AIM-V medium and seeded in T150 cm 2 flasks at 2.0 x 10 8 cells per flask.
  • Monocytes were then selected from the mononuclear cell population by adhesion to sterile tissue culture flasks for one to two hours at 37°C, 5% CO 2 , ⁇ 75% humidity.
  • Nonadherent and semi-adherent cells were removed by gentle washing with PBS.
  • the remaining adherent cells which were predominantly monocytes, were cultured in X-VIVO 15 culture medium containing 1000 U/ml GM-CSF and 1000 U/ml IL-4. The cells were incubated for 6 days at 37°C, 5% CO 2 , ⁇ 75% humidity to allow differentiation of monocytes into immature dendritic cells.
  • the immature dendritic cell-rich population was harvested by tapping the flasks to dislodge the cells. Cells in suspension were transferred to conical tubes. PBS was added to the flasks to remove the remaining floating cells and residual medium, and then added to the cell suspension in the same conical tubes. Detachment of remaining adherent immature dendritic cells was promoted by incubation in PBS at 2-8 °C. At the end of the incubation period, the flasks were tapped and the contents added to the cell suspension in the same conical tubes. The total cell suspension was then pelleted and resuspended in PBS, and a sample was removed to determine the cell concentration.
  • Immature dendritic cells were washed and resuspended in ViaSpan and transfected with 2 ⁇ g antigen-encoding mRNA per 10 6 dendritic cells. Electroporation was performed in 4 mm gap cuvettes containing 0.4 ml of the cell suspension (4 x 10 7 cells/ml) at a pulse of 300V, 100 ⁇ and 150 ⁇ F.
  • transfected cells were diluted with X-VIVO 15 medium, centrifuged and resuspended in X-VIVO 15 medium (serum free) supplemented with GM-CSF (800 U/ml), IL-4 (500 U/ml), IL-1 ⁇ (10 ng/ml), IL-6 (150 ng/ml), TNF- ⁇ (10 ng/ml) and PGE 2 (1 ⁇ g/ml).
  • GM-CSF 800 U/ml
  • IL-4 500 U/ml
  • IL-1 ⁇ 10 ng/ml
  • IL-6 150 ng/ml
  • TNF- ⁇ 10 ng/ml
  • PGE 2 (1 ⁇ g/ml
  • Transfected mature dendritic cells were suspended in heat-inactivated autologous plasma and 10% dextrose, at a final concentration of 4 x 10 7 cells/ml. The cells were then diluted 1:1 with a mixture of 20% DMSO to give a final concentration of 2 x 10 7 cells/ml in heat-inactivated autologous plasma which contains 10% DMSO and 5% dextrose, and then frozen in sterile cryovials at ⁇ 150 °C.
  • the data in this example supports the functionality and feasibility of producing RNA transfected dendritic cells from day-old apheresis product.
  • Dendritic cells were prepared by the method described in Example 3. The data below shows that dendritic cells can be reproducibly manufactured at the appropriate yield from a single day-old apheresis product and that the resulting cells (1) exhibit a classical mature phenotype, (2) can be efficiently transfected with RNA, and (3) can be cryopreserved with high post-thaw viability.
  • Immunophenotype of DCs The mature DCs were extensively characterized by FACS staining for molecular markers that should be present or absent from the final cell preparation.
  • HLA-DR, CD83, CD86, CD80, CD1a, and CD209 should show high expression and CD14, CD56, CD19, and CD3 should show low expression.
  • Table 3 shows the results (mean and standard deviation for percent positive cells) compiled from 11 consecutive runs of producing dendritic cell vaccines from PBMCS obtained from different healthy donors.
  • Table 3 Expression of Cell Surface Markers in DCs produced from Day Old Monocytes Marker % positive cells (Mean) % positive cells Standard Dev.
  • RNA-transfected mature dendritic cells Yield, phenotype and viability.
  • the dendritic cell methods have been thoroughly tested and shown to reproducibly generate high-quality RNA-transfected mature dendritic cells.
  • Table 4 shows the outcome of 11 vaccine runs using total amplified tumor cell line RNA as the antigen payload and normal donor dendritic cells as the vehicle.
  • RNA-transfected DCs express CCR7 and are migratory. In addition to the mature DC markers described above, CCR7 expression, which is critical for lymph node migration of the DCs in vivo, was evaluated. In this study FACS analysis with a CCR7-specific antibody was used to examine thawed RNA-transfected DCs produced using the full-scale GMP process (consistency runs 3, 4, 6, 7, 8, and 10).
  • RNA-transfected DCs provide functional costimulatory support.
  • the experiments described above show that the manufactured DCs express all of the critical costimulatory markers, while the experiments below demonstrate that these molecules are functional.
  • the ability of the DCs to stimulate interferon gamma (IFN- ⁇ ) production from PBMCs in an allo mixed lymphocyte (MLR) assay was determined using thawed DCs manufactured from 3 different donors along with previously frozen PBMCs from each donor. The expectation was that DCs would not stimulate INF- ⁇ production from their HLA-matched autologous PBMCs but would do so when mixed with non-autologous PBMCs. All pair wise combinations were tested using ELISPOT (INF- ⁇ ) as the readout. The data is presented in Figure 2 . The result of this experiment demonstrates that, as anticipated, only mismatched DC/PBMC combinations elicit INF- ⁇ production, a property which requires expression of functional MHC and costimulatory molecules.
  • Matured DCs have added functionality .
  • the cytokine cocktail used to mature the DCs post-transfection and pre-freeze contains TNF- ⁇ , IL-1 ⁇ , IL-6, and PGE 2 .
  • TNF- ⁇ IL-1 ⁇
  • IL-6 IL-6
  • PGE 2 PGE 2
  • T H 1 cytokine production from autologous T cells Both populations of DCs were transfected with RNA encoding Flu matrix protein and used to stimulate Flu-specific memory CTL from autologous PBMCs.
  • Figure 3 below shows the results of the ELISPOT analyses (# spots/well as a function of input PBMC).
  • IL-2 induction is considered to be an important because (1) induced IL-2 secretion sustains autocrine antigen-specific CTL proliferation and (2) low production of INF- ⁇ and IL-2 has recently been shown to correlate with increased mortality risk in HIV patients and in a recent study, lack of secretion of INF- ⁇ or IL-2 has resulted in impaired T cell function and an inability to maintain central memory responses. For simplicity, the negative controls were not graphed.
  • the average number of spots observed from the PBMC alone i.e., no DCs was 9.7 (INF- ⁇ ) and 1.3 (IL-2).
  • This experiment was repeated with vaccines made from the PBMCs of 3 independent donors and the results were qualitatively identical. Accordingly, the mature DCs have added and superior functionality compared to the immature DCs.
  • Freeze-thaw does not affect DC function .
  • the functionality of DCs pre-freeze and post-thaw was compared. Functionality was assessed by the ability of the DCs to stimulate a memory Flu-specific response from autologous PBMC as a function of decreasing Flu mRNA concentration used for transfection.
  • the assay readout was ELISPOT (INF- ⁇ ). Results are shown in Figure 5 below and indicate that the freeze-thaw process has no effect on the functionality of the DCs in this assay.
  • a constant amount of GFP mRNA (0.5 ⁇ g) was mixed in to monitor transfection efficiencies. Post-thaw samples were thawed after being frozen for 24 hours.
  • PBMCs PBMCs obtained by leukapheresis from healthy volunteers were enriched for monocytes by adherence to plastic flask following a 2-hour incubation in vitro. After washing, adherent cells were cultured for six days in X-VIVO 15 medium supplemented with recombinant granulocyte/macrophage-colony stimulating factor (rGM-CSF) and interleukin-4 (rIL-4) to generate immature DCs (iDCs).
  • rGM-CSF granulocyte/macrophage-colony stimulating factor
  • rIL-4 interleukin-4
  • iDCs were then electroporated (300V, 150 ⁇ F, 100 ⁇ ) with RNA encoding viral or control proteins and induced to mature for 24 hours in X-VIVO 15 medium supplemented with IL-1 ⁇ , IL-4, IL-6, GM-CSF, TNF- ⁇ and prostaglandin E 2 (PGE 2 ).
  • DCs were electroporated with RNA encoding the Green Fluorescent Protein (GFP) and expression was measured by flow cytometry. As shown in Figure 6 , a large fraction of mature DCs produced from day-old monocytes expresses high levels of GFP up to four days after transfection, confirming that this method is efficient at promoting long-term protein expression in DCs.
  • GFP Green Fluorescent Protein
  • CMV-infected subjects were identified by stimulating PBMCs from several blood donors with well-defined immunodominant peptides derived from the pp65 protein and measuring IL-2/IFN- ⁇ secretion as well as cell proliferation in both CD4 and CD8 T cells. Individuals in which positive CMV-specific T cell responses were detected and who agreed to undergo leukapheresis after informed consent were selected for further studies.
  • DCs from these individuals were prepared as described above. Mature DCs transfected with RNA encoding the CMV pp65 protein were then incubated for 16 hours (ICS) or 6 days (proliferation) with autologous PBMCs at a 1/40 ratio. After stimulation, IL-2 and IFN- ⁇ secretion ( Figure 7 ) and proliferation ( Figure 8 ) of CMV-specific CD4 and CD8 T cells was assessed by flow cytometry. DCs electroporated with CMV pp65 RNA selectively induced high IFN- ⁇ and IL-2 expression as well as proliferation of CD8 T cells from CMV-infected subjects. However, this protocol induces minimal CD4 T cell activation, as shown by the low level of cytokine secretion and proliferation detected in CD4 T cells ( Figures 7 and 8 , lower panels).
  • PBMC's from day-old leukapheresis maintained at a temperature of 6-28°C were washed and seeded @ ⁇ 2x10 8 / flask in AIM-V media for a 2 hour adherence step. After 2 hours, non-adherent cells were removed, and adherent cells were washed and resusupended in X-VIVO 15 supplemented with 1000 U/ml GM-CSF and either 500 U/ml or 1000 U/ml IL-4; incubated @ 37°C for 6 days.
  • the cells were resuspended in fresh X-VIVO 15 medium supplemented with 1000 U/ml GM-CSF and 500 U/ml IL-4; the media and cells added back into flasks still containing the adherent cells; and the flasks were incubated @ 37°C for a further three days.
  • DCs were transfected with 2 ⁇ g GFP mRNA per 10 6 cells (20 ⁇ g GFP mRNA per 5x10 6 cells), resuspended in X-VIVO 15 and seeded at 1x10 6 /ml; supplemented with 800 U/ml GM-CSF and 500 U/ml IL-4 and matured with cytokine cocktail (TNF ⁇ - 10ng/ml; IL-1 ⁇ - 10ng/ml; IL-6 -100 ng/ml; PGE 2 - 1 ⁇ g/ml). The DC's were incubated overnight @ 37°C; 5% CO 2 .
  • Phenotyping was undertaken on immature DC's on day 6 (immature DC; Figure 9 ) and 24 hours post transfection (mature DC; Figure 10 ).
  • the yield (% Rec) and viability (% V) for each culture condition immediately post transfection and at 24 hour post transfection is shown in Tables 6A-C.
  • Table 6A 1000 U/ml IL-4 DC yield day 6 % Re c % V DC per Tn DC @0hr post Tn % Rec % V DC @24hr post Tn % Rec % V F1 3.76x10 7 21 97 5x10 6 2.08x10 6 42 81 1.03x10 6 21 84 F2 3.88x10 7 22 94 5x10 6 2.41 x10 6 48 84 8.80 x10 5 18 91 F3 4.44x10 7 25 96 5x10 6 2.06 x10 6 41 83 8.10 x10 5 16 84 pooled 5 x10 7 1.04 x10 7 52 80 7.40 x10 6 37 94 pooled 2 x10 7 1.11 x10 7 56 93 7.50 x10 6 38 93 Table 6B 500 U/ml IL-4 DC yield day 6 % Rec % V DC per Tn DC @0hr post Tn % Rec % V DC @24hr post Tn % Rec % V F
  • Elutriation also known as counter-flow centrifugation, was performed on the ElutraTM Cell Separation System (Gambro BCT, Lakewood, CO) as an automated method to isolate monocytes from a day old leukapheresis, which had been shipped to the manufacturing facility from a collection site by overnight delivery in a temperature controlled (6-28°C) container.
  • Elutriation buffer was prepared by adding 1000mL of 5% Human Albumin Serum (HSA, Baxter Healthcare, Westlake, CA) to a 4L bag of Hank's Balanced Salts Solution (HBSS, Cambrex Bio Science, Walkersville, MD).
  • HSA Human Albumin Serum
  • HBSS Hank's Balanced Salts Solution
  • This elutriation buffer was added to the day old pheresis product in a volume equal to that of the pheresis.
  • the ElutraTM Cell Separation System (Gambro BCT, Lakewood, CO) was primed with elutriation buffer and the pheresis product was loaded. After the elutriation procedure was performed, monocytes were collected from the rotor off fraction. The monocytes were stored frozen.
  • iDC immature dendritic cells
  • mDC mature dendritic cells
  • Peripheral blood was collected from 3 healthy human donors on three separate days by leukapheresis and transported to the University of Montreal within 30 minutes post collection. Twenty mL of the leukapheresis was removed and autologous plasma was prepared by centrifugation. The leukapheresis volume was divided into two equal parts. One part was processed immediately to generate "fresh" monocytes while the second part was stored as 20 mL aliquots in five 50 mL conical tubes to generate cells from a "day old pheresis". The tubes were placed in a plastic container within a box that was stored tilted on a rocking platform between 16-20°C for 24 hours. Following the incubation period, the PBMCs were separated using Ficoll density gradient.
  • a mononuclear cell fraction that includes the dendritic cell precursors (monocytes) was separated from both fresh and day old pheresis using a Ficoll density gradient.
  • the Leukapheresis was layered onto Ficoll in 50 mL conical tubes and tubes were centrifuged (800Xg) for 20 minutes at room temperature. Cells were washed four times with phosphate buffered saline (PBS), and counted to determine cell concentration and cell viability.
  • PBS phosphate buffered saline
  • CD14 microbeads CD14 microbeads
  • 1x10 9 cells were resuspended in 8 mL of buffer containing PBS, 0.5% BSA, and 2 mM EDTA (Miltenyi buffer). 2 mL of CD14 microbeads were added to each 50 mL tube containing the 1x10 9 cells and incubated at 4°C for 15 minutes. The cells were washed in 100 mL of the same buffer, centrifuged at 300Xg and resuspended in 20 mL of Miltenyi buffer. The cell suspension was applied to four LS columns situated under magnetic field (Miltenyi QuadromaxTM). Following application of the cells using gravity flow, the columns were washed three times with 3 mL of Miltenyi buffer.
  • the monocytes were eluted twice in the absence of a magnetic field with 5 mL of Miltenyi buffer and centrifuged at 300Xg for 10 minutes. Purity of both the eluate and flow through fractions were determined with flow cytometry using antibodies specific for CD3, CD19, CD16, CD56, CD14, and CD209. The eluate fraction contained 88-98% monocytes and less than 2% small cells. The nonadherent fractions contained only a small percentage (2 %) of monocytes. Total RNA was extracted from a portion of the purified monocytes (50 million) at this time.
  • TIP-DCs To prepare mature differentiated DCs, 50 million monocytes were seeded into T150cm 2 flasks in X-VIVO medium containing IL-4 and GM-CSF at 37°C for five days. A cytokine cocktail (TIP) containing tumour necrosis factor, interferon gamma, and prostaglandin E2 was added to the cells on day five. Cells were harvested on day six of culture and at this time were termed "TIP-DCs".
  • TIP-DCs cytokine cocktail
  • the dendritic cell-rich population was harvested by gently tapping the flasks to dislodge the cells, additional phosphate buffered saline (PBS) washes and detachment of the remaining adherent cells by incubating in PBS and at 2-8°C for approximately 10 minutes.
  • PBS phosphate buffered saline
  • the total cell suspension was pelleted and resuspended in PBS, and analyzed for cell concentration, cell viability and immunophenotyping.
  • monocyte lineage markers CD3, CD14, CD19, CD16, and CD56
  • indication of presence of dendritic cells CD11c, CD1a, and CD209
  • an antigen presenting cell marker HLA-II
  • markers of migration CD38 and CCR7
  • markers for mature dendritic cells CD83 and CD86.
  • PME-CD40L DCs were frozen in 90% autologous plasma with 10% DMSO, in cryovials using controlled-rate freezing, and stored at ⁇ -85°C.
  • a frozen dendritic cell vaccine prepared as described above was thawed at 37°C, and kept at 20-25°C for 30 minutes. Viability was determined immediately post-thaw and at 10 minute intervals up to 30 minutes.
  • Post Thaw PME-CD40L DCs were analyzed by flow cytometry using antibodies specific for monocyte lineage markers (CD 14), dendritic cell markers (CD11c, CD1a, and CD209), an antigen presenting cell marker (HLA-II), markers of migration (CD38 and CCR7) and markers for mature dendritic cells (CD80, CD83 and CD86).
  • the data from the three donors demonstrates that positive selection using CD14 bead results in 88-98% pure population of monocytes (Table 7).
  • the maximum amount of small cell contamination is 2% (Table 7).
  • the nonadherent (flow through) fraction contained up to 2% of monocytes (large cells) in all three donors (data not shown). Therefore, there is no significant loss of monocytes in the nonadherent fraction.
  • the small cell population contamination present in the eluate is composed primarily of T cells as evident from high CD3 expression and lower CD19, 16 or 56 markers expression (data not shown). There were no visible differences in expression of CD83, CD86, CD11C, HLA-I, or HLA-II markers in the eluate fraction between fresh and day old leukapheresis.
  • the percentage of positive cells refers to the number of cells positive for a marker under investigation.
  • the percentage of DCs expressing the surface maturation marker CD83 was lower in TIP-DCs from all 3 donors prepared from fresh leukapheresis than those prepared from day old leukapheresis (Table 8).
  • PME-CD40L DCs The DCs post-transfection (PME-CD40L DCs) were analyzed for expression of CD 154 (CD40L) four hours after transfection.
  • PME-CD40L DCs from day old leukapheresis expressed more CD40L in donors 1 and 2 four hours after transfection than PME-CD40L DCs generated from fresh leukapheresis (Table 9).
  • MFI mean fluorescence intensity
  • PME-CD40L DCs were analyzed for expression of a variety of DC markers after thawing. There were differences in the phenotype of the post thaw PME-CD40L DCs generated from fresh versus day old leukapheresis. The mean fluorescence intensity of CD40L expression in PME-CD40L DCs prepared from fresh leukapheresis was lower than DCs prepared from day old Leukapheresis (Table 9).
  • RNA samples from fresh monocytes and day old monocytes (monocytes which had been incubated at 16-20°C for 24 hours following leukapheresis), and RNA from DCs prepared from fresh and day old monocytes, as described in Example 8, were applied to the Human Genome U133 Plus 2.0 Array (Affymetrix, Santa Clara, Calif.) according to the manufacture's instruction (Genechip ® Expression Analysis Technical Manual, 2004). Briefly, three micrograms of total RNA spiked with Genechip ® Poly-A RNA Control Kit (Affymetrix, Santa Clara, Calif.) was converted to first-strand cDNA using SuperScriptTM II reverse transcriptase.
  • Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP.
  • the cRNA products were fragmented to around 100 nucleotides, and hybridized for 16 hours to the microarrays.
  • the microarrays were then washed at low (6XSSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin.
  • the GeneChip ® Scanner 3000 (Affymetrix, Santa Clara, Calif.) was used to collect fluorescence signal at 3 um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature. Scanned images were analyzed with Genechip ® Operating Software v1.1 (Affymetrix, Santa Clara, Calif.). High linear correlation (R 2 >0.95) of 4 control RNAs included in Poly-A RNA Control Kit (Affymetrix, Santa Clara, Calif.) was confirmed to guarantee the success of the labeling process.
  • the data was first filtered for flags with missing spots.
  • the data was then analyzed with a one way anova without random prediction models with a confidence level ranging from p.05 or p. 1.
  • the list of filtered genes generated was then analyzed for either fold changes, level of expression, or confidence. This was performed using the samples as averages or as individual samples.
  • Level of expression between fresh and day monocytes or dendritic cells were compared prior to or after an anova.
  • the lists of genes were compared to one another and those overlapping in several lists were chosen for their reliability. Genes with altered steady state RNA levels in dendritic cells prepared from day old monocytes versus dendritic cell prepared from fresh monocytes are listed in Table 12A. Descriptions of these genes are listed in Table 12B.
  • Table 12A Genes with Altered Steady State RNA Levels in Dendritic Cells prepared from Day Old Monocytes as compared to Dendritic Cells Prepared from Fresh Monocytes Gene Symbol Affymetrix Identifier NCBI Unigene Fold P value Change in Day Old ALOX15 207328_at NM_001140 Hs.73809 5.8 0.0699 Decrease 1558517_s_at AK094804 Hs.24181 5.5 0.0957 Decrease KRT17; PC; K17; PC2; PCHC1 212236_x_at NM_000422 Hs.2785 5 0.0759 Decrease KCNJ2 231513_at, 206765_at NM_000891 Hs.1547 4.5 0.0507 Decrease YWHAZ 214848_at NM_003406, Hs.492407 4.3 0.0581 Decrease NM_145690 IL1B 205067_at, 39402_at NM_000576 Hs.126
  • the dendritic cell genes were further normalized to expression of two housekeeping genes shown to remain constant between DCs produced from fresh and day old monocytes.
  • the average expression of each gene (normalized) was divided by the average expression (normalized) of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) or ⁇ -actin to produce a ratio of expression relative to the housekeeping gene.
  • GAPDH glycosylase dehydrogenase
  • ⁇ -actin ⁇ -actin
  • TaqMan ® Gene Expression Assays or Custom TaqMan ® Gene Expression Assays were used as TaqMan ® probes. PCR reactions were performed in triplicate using ABsoluteTM QPCR ROX Mix (ABgene, Surrey, UK). Quantitation of relative cDNA concentrations was done using the relative standard curve method as described in ABI Prism ® 7700 Sequence Detection System User Bulletin #2 (Applied Biosystems, Foster City, Calif.). One cDNA with the most expression of each gene in the GeneChip analysis was used for generating the relative standard curve. All data are shown as expression relative to internal GAPDH expression.
  • APAF-1 and CDCA2 effector protein 2 were demonstrated to decrease, relative to GAPDH, at least 2.5 fold and 3 fold, respectively between fresh and day old monocytes.
  • Table 15 Primers Gene TaqMan® Gene Expression Assays ID GAPDH Hs99999905_m1 CDC42 effector protein 2 Hs00198943_m1 APAF1 Hs00185508_m1
  • PBMCs leukopheresis product
  • PBMCs were prepared by ficoll-histopaque density centrifugation, and washed four times in PBS at room temperature. 2x 10 8 PBMCs were re-suspended in 30ml AIM-V medium (Invitrogen) and allowed to adhere to 150 cm 3 plastic flasks for 2 hours at 37°C. Non-adherent cells were removed and remaining cells cultured in X-VIVO 15 medium, supplemented with 1000 U/ml GM-CSF (Leukine) and 1000 U/ml IL-4 (R&D systems), for 5 days at 37°C, 5% CO 2 .
  • AIM-V medium Invitrogen
  • Immature DCs were initially matured by addition of TNF- ⁇ (10 ng/ml), IFN- ⁇ (1000 U/ml) and PGE 2 (1 ⁇ g/ml). After overnight culture, the TIP-DC intermediate products were harvested by removal of media, and a cold PBS wash. TIP-DC were phenotyped to confirm the generation of a mature population of cells. To generate antigen-loaded, fully mature DC, the TIP-DC were electroporated: Cells were re-suspended in chilled Viaspan at 4x10 7 /ml in 0.5 ml and placed on ice.
  • DCs were mixed with amplified total tumor renal cell carcinoma mRNA at 1 ⁇ g/10 6 cells, as a model antigen-encoding payload, plus 4 ⁇ g/10 6 CD40L mRNA, and placed in a 4 mm gap electroporation cuvette, followed by electroporation using a Biorad GenePulsar Xcell system. Immediately after electroporation, DCs were washed in X-VIVO 15 medium and finally re-suspended in 20 ml of X-VIVO 15 supplemented with GM-CSF (800 U/ml) and IL-4 (500 U/ml) at 1x10 6 /ml, and cultured for 4 hours at 37°C in low adherence T75 flasks.
  • GM-CSF 800 U/ml
  • IL-4 500 U/ml
  • PBMCs 3x10 5 cells were each stained with CD19-PE, CD14-PE and CD3-PE, or matched isotype conjugated controls by incubation in antibody for 30 minutes at 4C. After incubation, the stained cells were washed 3 times in cold 1% FBS/PBS and resuspended in PBS for fluorescence analysis using a FACScalibur flow cytometer (BD Biosciences) using CellQuest software (BD Biosciences). 3 minutes prior to acquisition, propidium iodide (1ug/ml) was added to each sample as a viability dye for gating purposes.
  • DCs 1x10 6 cells (TIP-DC and PME-CD40L DC) were re-suspended in chilled PBS/1% FBS.
  • Intracellular CD40L was determined as follows: 2 x 10 5 PME-CD40L DCs re-suspended in 250 ⁇ L of Cytofix/Cytoperm solution (BD Biosciences) for a minimum of 10 minutes up to 2 hours at 4°C. Cells were washed twice with 2ml staining buffer (PBS, BSA, NaN 3 , and EDTA), re-suspended in 0.5 ml staining buffer and stored over night at 4°C. Cells were re-suspended in 2.0 ml Perm/Wash solution (BD Biosciences) for 15 minutes, centrifuged and re-suspended in 100 ⁇ l Perm/Wash solution.
  • 2ml staining buffer PBS, BSA, NaN 3 , and EDTA
  • mice anti-human CD40L APC or mouse IgG1 APC was added to each DC preparation and incubated at 4°C for 30 minutes in the dark. Cells were washed twice with 1ml Perm/Wash solution and re-suspended in staining buffer prior to flow cytometric analysis. The results are shown in Table 18.
  • Tables 17 and 18 show that TIP-DCs generated from the various starting products are equally amenable to electroporation, and recovery, with full maturation into the final product, PME-CD40L DC.
  • PME-CD40L DC generated from this study can be formulated as 'vaccine' and frozen and thawed without any deterioration of the DC preparations.
  • apheresis products held for up to 72hrs post collection are a viable raw material for centralized manufacturing of DC vaccines for application in the clinic.

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